The central nervous system is a major target for alcohol and its consumption has long been associated with brain damage. Ethanol may have several cellular targets in different brain areas, where ethanol may influence glutamatergic excitatory transmission. In this study we have used rat hippocampal astrocytes in culture to investigate the effect of ethanol on kainate-induced glutamate secretion and on intracellular free Ca2+ concentration ([Ca2+]c). Cell cultures were prepared by digestion of hippocampi with papain, followed by mechanical disgregation, employing previously described methods (González et al., 2006). Astrocytes were cultured in medium consisting of DMEM supplemented with foetal bovine serum (10%), penicillin (20000 IU), streptomycin (20 mg/mL) and MITO+ (0.1%), at 37 °C in a humidified incubator (5% CO2). Glutamate release was monitored using an enzyme-linked system by which, in the presence of glutamate, glutamate dehydrogenase reduces NADP+ to NADPH. NADPH fluoresces when excited with UV light (Rodriguez-Moreno and Sihra, 2004). [Ca2+]c was monitored by single cell imaging analysis of fura-2-loaded cells. Our results show that kainate (10 µM to 500 µM) stimulated glutamate release from astrocytes. Preincubation of astrocytes in the presence of ethanol (1 mM-50 mM) induced a concentration-dependent inhibition of glutamate release caused by stimulation of cells with 100 µM kainate. Inhibition of alcohol-dehydrogenase, by preincubation of astrocytes in the presence of 4-methylpyrazole (1 mM), abolished ethanol-induced inhibition of glutamate release in response to kainate. On the other hand, preincubation of astrocytes in the presence of the antioxidant cinnamtannin B-1 (10 µM), also blocked ethanol inhibitory action on glutamate release in response to kainate. Stimulation of astrocytes with kainate (100 µM) induced an initial increase in [Ca2+]c followed by a progressive decrease. Ethanol (50 mM) reduced Ca2+ mobilization in response to kainate, whereas cinnamtannin B-1 reversed the inhibitory action of ethanol on Ca2+ mobilization by kainate. Our results are consistent with an inhibitory action of ethanol on glutamate secretion from hippocampal astrocytes. The inhibitory action of ethanol is probably due to its oxidative metabolization, and may involve reactive oxygen species production and a lower Ca2+ mobilization by kainate. Taking into account the pivotal role that astrocytes play within the central nervous system, especially in relation to neurons, the negative effects of ethanol on the release of glutamate might affect neuron-glia communication in the hippocampus, which might lead to functional defects in the brain.